Differential inflammation responses determine the variable phenotypes of epilepsy induced by GABRG2 mutations

Abstract Objective To explore the mechanism involved in variable phenotypes of epilepsy models induced by γ‐aminobutyric acid type A γ2 subunit (GABRG2) mutations. Methods The zebrafish carrying wild‐type (WT) GABRG2, mutant GABRG2(P282S), GABRG2(F343L) and GABRG2(I107T) were established by Tol2kit transgenesis system and Gateway method. Behavioral analysis of different transgenic zebrafish was performed with the DanioVision Video‐Track framework and the brain activity was analyzed by field potential recording with MD3000 Bio‐signal Acquisition and Processing System. The transcriptome analysis was applied to detect the underlying mechanisms of variable phenotypes caused by different GABRG2 mutations. Results The established Tg(hGABRG2 P282S ) zebrafish showed hyperactivity and spontaneous seizures, which were more sensitive to chemical and physical epileptic stimulations. Traditional antiepileptic drugs, such as Clonazepam (CBZ) and valproic acid (VPA), could ameliorate the hyperactivity in Tg(hGABRG2 P282S ) zebrafish. The metabolic pathway was significantly changed in the brain transcriptome of Tg(hGABRG2 P282S ) zebrafish. In addition, the behavioral activity, production of pro‐inflammatory factors, and activation of the IL‐2 receptor signal pathway varied among the three mutant zebrafish lines. Conclusion We successfully established transgenic zebrafish epileptic models expressing human mutant GABRG2(P282S), in which CBZ and VPA showed antiepileptic effects. Differential inflammatory responses, especially the SOCS/JAK/STAT signaling pathway, might be related to the phenotypes of genetic epilepsy induced by GABRG2 mutations. Further study will expand the pathological mechanisms of genetic epilepsies and provide a theoretical basis for searching for effective drug treatment.


| INTRODUC TI ON
Mutations in γ-aminobutyric acid type A (GABA A ) receptor subunit genes (GABRs) are frequently associated with various phenotypes of epilepsy, from mild epileptic syndromes such as childhood absence epilepsy (CAE) and febrile seizures (FS) to more severe epileptic phenotype such as Dravet syndrome (DS), Lennox-Gastaut syndrome (LGS), and West syndrome (WS). 1,2GABA A receptor is a chloride channel mediating most of the fast inhibitory neurotransmission in the central nervous system (CNS), which is usually composed of five subunits, two α1 subunits, two β2 subunits, and one γ2 subunit (2α12β2γ2).γ2 subunit (encoded by GABRG2) plays an important role in GABA A receptor functions and mutations of γ2 subunit often induce the occurrence of epilepsy. 3Although some studies have investigated the relationship between genotypes and phenotypes of epilepsy induced by different GABRG2 mutations, 4 the mechanisms remain unclear.
The structure of γ2 subunit contains four transmembrane domains (TM1 to TM4) and a long extracellular N-terminal domain.It was reported that the phenotype was more related to the location of variants within the protein and the variants in transmembrane domains had the most severe phenotype in terms of epilepsy. 4Our previous study has found mutations located in different structural domains leading to epileptic encephalopathies. 5To further investigate the complicated relationship between phenotypes and genotypes, we have established zebrafish epilepsy models overexpressing different mutant human GABRG2, including the F343L mutation located in the TM3 6 and the I107T mutation located in the N-terminal.These zebrafish models displayed spontaneous seizure activity and convulsive behaviors at the larval stage.Compared with rodent models, the shorter maturation period of zebrafish provides a better platform for investigating the mechanisms of epilepsy with different genotypes and screening of antiepileptic drugs (AEDs).The establishment of the above zebrafish epilepsy models with GABRG2 mutations facilitated the study of molecular mechanisms involved in the relationship between genotypes and phenotypes of epilepsy, thus promoting the development and clinical application of specific AEDs.
Previous research has indicated that neuroinflammation is involved in epileptogenesis. 7We have also demonstrated that neuroinflammation was induced in febrile seizures, and the increased pro-inflammatory factors, such as interleukin 1β (IL-1β) and IL-6, might be responsible for the epileptogenesis in fever-associated epilepsy. 8Increased pro-inflammatory cytokines including tumor necrosis factor-alpha (TNFα), IL-1β, and IL-6 were also observed in an epilepsy mouse model with Gabrg2 +/Q390X knockin, indicating neuroinflammation was one of the mechanisms for genetic epilepsy. 9wever, whether neuroinflammation is involved in other phenotypes of epilepsy induced by GABRG2 mutations and is related to the severity of epilepsy needs further exploration.
In this study, we first established a new transgenic zebrafish line carrying mutant human GABRG2(P282S), which occurred in the TM1 and observed its epileptic phenotype.Then we tested some traditional and new AEDs in this epilepsy model to find out effective AEDs.In addition, we compared the behavior characters among three different mutant GABRG2 transgenic zebrafish as well as the transcriptome changes to further understand the underlying mechanisms involved in variable phenotypes induced by different GABRG2 mutations.

| Behavioral analysis
The swimming movements of the zebrafish larvae at 5 days postfertilization (dpf) were recorded with the DanioVision Video-Track framework (Noldus, German) for 30 min after 30 min adaption in the recording chamber.The zebrafish induced with 15 mM PTZ were also recorded for 30 min right after the stimulation.The zebrafish stimulated with light flash stimulation (light on for 5 s and turn off for 5 s) were recorded for a total of 100 cycles.All the data were analyzed with the EthoVision XT locomotion tracking software (Noldus, Germany).The average distance traveled, the activity of mobility, and the time spent in movement for each group were calculated and compared.

| Whole-mount in situ hybridization (WISH) and the quantitative reverse transcription polymerase chain reaction (qRT-PCR)
The mRNA expressions of c-fos in the brains of zebrafish were measured with WISH and qRT-PCR.The WISH was performed with the modified Thisse's method as previously reported. 6qRT-PCR was performed with SYBR green mix (Vazyme, China) after RNA extraction with TRIzol reagent (Invitrogen, USA) and cDNA transcription with an Omniscript RT kit (Qiagen, USA) according to the manufacturer's protocols.The sequences of primers used are listed in Table S1.β-actin was used as the internal control.Data were normalized to the internal reference gene β-actin and quantified relative to

| Field potential recording
The field potential recording of zebrafish was performed with the MD3000 Bio-signal Acquisition and Processing System (Anhui Zhenghua, China).In brief, the zebrafish were embedded in recording media solution (1 mM NaCl, 2.9 mM KCl, 10 mM HEPES, 1.2 mM MgCl 2 , 10 mM Dextrose, 2.1 mM CaCl 2 ) with 1.2% low melting agarose and 0.02% tricaine to anesthetize the animals.The field potential recording was performed for each zebrafish with electrodes inserted into the forebrain.The diaphragm clamp amplifier was set to a magnification of 1000, an upper limit of 10,000 Hz, a time constant of 0.5 ms, and a high-pass filter of 100 Hz.

| Transcriptomic assay
The brain samples underwent RNA extraction using TRIzol Reagent and 1 μg total RNA with a RIN value above 6.5 was used for the following library preparation and transcriptome sequencing using the HiSeq Control Software (HCS) + OLB + GAPipeline-1.6(Illumina) on the HiSeq instrument.Gene sets with a p < 0.05 and a false discovery rate <0.05 were considered significantly enriched genes.Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were conducted as previously reported. 10e protein-protein interaction (PPI) network of the differentially expressed genes (DEGs) was established with the STRING database (https:// cn.strin g-db.org/ ).The data analysis was processed by GENEWIZ (Suzhou, China).

| Statistical analyses
Data are expressed as mean ± SEM.Statistical analysis was performed using GraphPad Prism 8.0 (GraphPad, San Diego, CA, USA).
The normality of all data was analyzed by Kolmogorov-Smirnov test or Shapiro-Wilk test.Unpaired Student's t-test or one-way ANOVA and subsequent Tukey's multiple comparisons test were used to compare the differences between groups when the data exhibited normal distribution.Mann-Whitney U-test or Kruskal-Wallis test and subsequent Dunn's multiple comparisons test were used when the data did not conform to a normal distribution.Statistically significant was set as p < 0.05.

| The establishment of Tg(hGABRG2 P282S ) zebrafish with hyperactivity and spontaneous seizures
To generate transgenic zebrafish with neuronal specifically expressing WT GABRG2 or mutant GABRG2, we used pDestTol2CG2 plasmid with neuronal-specific HuC promoter (Figure S1A,B).The cmlc2: EGFP in the vector was to label the embryonic heart with enhanced green fluorescent protein (EGFP) for screening embryos with human GABRG2 expression.The recombinant plasmids were microinjected into one-cell stage zebrafish embryos with transposase mRNA and the embryos with EGFP-labeled hearts were screened out for further breeding (Figure S1C).The expression of human GABRG2 and the genomic sequence of transgenic zebrafish were confirmed with RT-PCR (Figure S1D) and Sanger sequencing (Figure S1E), showing the change of "C" to "T" at 844, which would translate from Proline (P) to Serine (S) at site 282 in the mutant group.The above results suggested that we successfully established transgenic zebrafish carrying human WT GABRG2 or mutant GABRG2(P282S).
Locomotor activities monitored with a video tracking system (Noldus, German) were used to investigate whether the expression of mutant GABRG2(P282S) would affect the neuronal hyperexcitability of the transgenic zebrafish.At 5 dpf, the representative swimming tracks of Tg(hGABRG2 WT ) and Tg(hGABRG2 P282S ) larvae were shown in Figure 1A.The distance traveled in the 30-min recording was plotted in Figure 1B and the total distance traveled in each group was quantified in Figure 1C.The swimming distance increased significantly in the P282S group (2592 ± 140 mm vs. 1899 ± 134 mm in the WT group, p = 0.0013).The proportion of P282S zebrafish in immobile activity decreased significantly (92.10 ± 0.46% in the P282S group vs. 93.96± 0.49% in WT group, p = 0.0033, Figure 1D) while the movement duration increased in the P282S zebrafish (599.6 ± 34.00 s vs. 432.3± 34.55 s in WT group, p = 0.0036,  Field potential recording in the forebrain was performed to observe the seizure activity in the zebrafish larvae.Multispike, largeamplitude, and long-duration burst discharges were detected in the P282S zebrafish (Figure 1G), with a significant increase of the maximal amplitude (0.075 ± 0.005 vs. 0.033 ± 0.002, p = 0.0011, Figure 1H).To further detect the brain activity in transgenic zebrafish, the transcription factor, c-fos, a hallmark of seizure onset in vertebrates, was measured with WISH and qRT-PCR.The qRT-PCR quantification analysis showed that the expression of c-fos mRNA in the P282S group was much higher than that in the WT control (3 dpf: 2.850 ± 0.096 vs. 1.000 ± 0.033, p < 0.0001; 5 dpf: 2.258 ± 0.142 vs. 1.000 ± 0.043, p < 0.0001, Figure 1I).WISH images showed that c-fos was expressed throughout the brain, including telencephalon, optic tectum, midbrain, and hindbrain at 3 and 5 dpf (Figure 1J).The above results suggested that P282S zebrafish was more active in the swimming behavior and had spontaneous seizures at the larval stage, indicating a successful establishment of a zebrafish epilepsy model carrying human mutant GABRG2 (P282S).
Frequent light flash stimulation is inducement of seizures in some patients with epilepsy.To detect whether the mutant P282S zebrafish also has a similar phenotype, we set the light flash stimulus condition with 100 cycles of 5 s light on and 5 s light off.The swimming trajectory is plotted in Figure 2E.Upon light stimulation, we noticed that both the acceleration maximum (Figure 2F) and total distance traveled in P282S larvae increased (898.8 ± 87.06 mm vs. 643.6 ± 56.15 mm in the WT group, p = 0.0138, Figure 2G), indicating a hyperactive response to light flash exposure.However, the mobile activity of P282S larvae was not significantly changed with light flash stimulation (Figure 2H).These results suggested that the P282S mutant zebrafish were more sensitive to chemical and physical stimulations which would induce epileptogenesis.

| CBZ and VPA ameliorated hyperactivity in Tg(hGABRG2 P282S ) zebrafish
As the patient carrying P282S mutation showed secondary generalized onset and remained intractable with slight improvement with lamotrigine (LTG) treatment, we screened some traditional AEDs in the Tg(hGABRG2 P282S ) zebrafish model to give a hint for further clinical application.Various drugs, including 100 μM CZP, 30 mM LEV, 100 μM CBZ, and 100 μM VPA, were added to the culture medium for 30 min at 5 dpf and the locomotor activity was recorded (Figure 3A).

| Comparison of different mutant GABRG2 transgenic zebrafish models
Several de novo GABRG2 pathogenic variants, including P282S, F343L, and I107T, have been reported to induce epileptic encephalopathy in patients. 5I107T occurred in the N-terminal domain, while P282S and F343L occurred in the TM1 and TM3 transmembrane domains, respectively.To detect the effect of locations of mutations on phenotypes, we compared the locomotive activity of different mutant GABRG2 transgenic zebrafish.In the previous research, we generated F343L and I107T transgenic zebrafish with spontaneous seizures. 6,10In this research, the distance traveled in 30 min at 5 dpf was compared in the above three transgenic lines and the data showed that all mutant zebrafish traveled more than the WT control group, showing an epileptic phenotype.Among the three mutant zebrafish models, the I107T line traveled the most while the P282S line traveled the least, and the difference between the two groups was significant (1774 ± 94.55 mm in the I107T group vs. 1125 ± 106.9 mm in the P282S group, p = 0.0010, Figure 4A), indicating a less active phenotype induced by P282S mutation.Previous research has found a broad-spectrum histone deacetylase (HDAC) inhibitor, SAHA, as an effective treatment for F343L and I107T zebrafish.Then we tried to detect whether it was also effective in the P282S zebrafish.
However, the data showed that SAHA did not reduce the travel distance in the P282S zebrafish (Figure 4A), further suggesting that the phenotypes were different in the three mutant transgenic zebrafish.

| Transcriptome changes in the brain of mutant GABRG2 transgenic zebrafish
To further investigate the mechanisms involved in P282S mutationinduced epilepsy, brain transcriptome was analyzed.At 3 dpf and 5 dpf, the brain tissues of WT and P282S zebrafish were collected for transcriptome sequencing.There was 269 (87 down-regulated and 182 up-regulated) and 1407 (428 down-regulated and 979 up-   S2 and S3. compared with the WT group at 3 dpf (Figure 5A, Table S2) and 5 dpf (Figure 5B, Table S3), respectively.
The KEGG pathway analysis showed that the number of DEGs related to metabolic pathways increased from 16 at 3 dpf to 136 at 5 dpf (Figure 5C,D).Among the DEGs at 5 dpf, 17 genes were related to peroxisome proliferator-activated receptors (PPARs), 14 genes were related to protein digestion and absorption, 14 genes were related to lipid digestion and absorption, and 15 genes were related to drug metabolism (Figure 5D).We validated the expression of some DEGs by qRT-PCR and the data showed that glutamate-ammonia ligase (glutamine synthase) c (glulc) and PET100 homolog (pet100) increased at both 3 dpf and 5 dpf, ubiquitin-conjugating enzyme E2D 4 (ube2d4), phoenix (pho), and neuronal PAS domain protein 4a (npas4a) decreased at both 3 dpf and 5 dpf, while shisa family member 9b (shisa9b) and choline Oacetyltransferase a (chata) increased at 3 dpf and decreased at 5 dpf (Figure 5E).The result was consistent with the transcriptome analysis.
Lastly, the difference in the brain transcriptome of the three mutant zebrafish lines was compared.A total of 27 genes were upregulated and 76 genes were down-regulated in all three lines at 3 dpf (Figure 6A), while 73 genes were up-regulated and 49 genes were down-regulated at 5 dpf (Figure 6B).The PPI network of the DEGs at both 3 dpf and 5 dpf was established with the STRING database, showing the involvement of interleukin-2 (IL-2) receptor signal pathway in the differential phenotypes of epilepsy caused by GABRG2 mutations (Figure 6C).The expressions of DEGs in the PPI network, including IL-2 receptor subunit beta (il2rb), IL-2 receptor subunit gamma a (il2rga), IL-2 receptor subunit gamma b (il2rgb), chemokine (C-X-C motif) receptor 3.1 (cxcr3.1),protein kinase C epsilon b (prkceb), protein kinase C eta a (prkcha), protein kinase cAMP-dependent catalytic beta a (prkacba), protein kinase cAMP-dependent catalytic beta b (prkacbb), calmodulin-like 4a (calml4a), fas cell surface death receptor (fas), cd40, signal transducer and activator of transcription 5a (stat5a), signal transducer and activator of transcription 5b (stat5b), and suppressor of cytokine signaling 1a (socs1a), were compared in the three transgenic lines at 3 dpf (Figure 6D) and 5 dpf (Figure 6E).Among the three transgenic zebrafish lines, the expressions of il2rb, il2rga, cxcr3.1,prkceb, prkcha, prkacba, calml4a, fas, cd40, stat5a and socs1a were the lowest in the I107T zebrafish, while il2rgb, prkacba and stat5b were the highest, especially at 3 dpf.On the other side, the changes of genes in the IL-2 receptor signal pathway in the P282S zebrafish were not as significant as that in the I107T zebrafish.The above data suggested the differential activation of the IL-2 receptor signal pathway might be related to variable phenotypes of epilepsy.

| DISCUSS ION
GABRG2(P282S) missense mutation was first detected in a patient with epileptic encephalopathy, who had secondary generalized seizure onset at 1 year old and followed by atypical absences. 5Although the symptoms showed a slight improvement with lamotrigine treatment, the seizures remained intractable even after combination therapy with AEDs. 5 To further investigate the potential mechanisms of genetic epilepsy induced by GABRG2 mutations, we established the transgenic zebrafish line carrying the GABRG2(P282S) mutation in vivo.We also compared different phenotypes and analyzed the brain transcriptomic changes of the epilepsy models induced by different GABRG2 mutations.Our data indicated that differential inflammation responses might determine the variable phenotypes of epilepsy induced by GABRG2 mutations.
In the previous research, we established zebrafish epilepsy models by expressing mutant human GABRG2(F343L) and GABRG2(I107T), which were discovered in patients.In this study, we constructed another epileptic zebrafish line with mutant GABRG2(P282S), exhibiting a similar phenotype in humans.The successful establishment of these animal models provided a reliable approach for studying the pathological mechanisms of epileptogenesis and exploring drug treatment.Some traditional AEDs, including CZP, LEV, CBZ, and VPA, were applied in the zebrafish model to find effective treatment for the patient.As CZP and LTG are both voltage-gated sodium channel blockers, 12,13 the ineffectiveness of CZP and LTG in animal models and patients indicated that sodium channels were not significantly affected in the epilepsy induced by GABRG2 mutations.5][16] Despite multiple molecular targets and mechanisms, the treatment with LEV did not show improvement of seizure activity in the mutant GABRG2(P282S) zebrafish model, further confirming the intractable phenotypes.On the other hand, CBZ and VPA showed a dose-dependent effectiveness in the GABRG2(P282S) zebrafish, which was not effective in the GABRG2(F343L) zebrafish.CBZ is one of the broad-spectrum AEDs against both generalized and focal seizures, which binds to GABA A receptors and influences the opening of chloride channel. 12Although CBZ is mainly used as an add-on treatment, it has been proven to be effective when used in monotherapy. 17VPA, one of the first line AEDs, which primarily enhances inhibitory neurotransmitter GABA, 13 also significantly reduced the activity of GABRG2(P282S) zebrafish.The different actions of these AEDs in the GABRG2(F343L) and GABRG2(P282S) zebrafish indicated F I G U R E 6 Differentially expressed genes in different mutant GABRG2 transgenic zebrafish.(A) Venn diagram of the up-regulated genes and down-regulated genes in the brains of P282S, F343L, and I107T transgenic zebrafish larvae at 3 dpf.(B) Venn-diagram of the upregulated genes and down-regulated genes in the brains of P282S, F343L, and I107T transgenic zebrafish larvae at 5 dpf.(C) PPI network of DEGs at both 3 dpf and 5 dpf.(D) The relative expressions of DEGs in the PPI network in the P282S, F343L, and I107T transgenic zebrafish larvae at 3 dpf.(E) The relative expressions of DEGs in the PPI network in the P282S, F343L, and I107T transgenic zebrafish larvae at 5 dpf.
the different mechanisms or severity of these transgenic models.
Not similar to GABRG2(F343L) and GABRG2(I107T) zebrafish, the HDACs inhibitor SAHA did not rescue the epileptic phenotype of GABRG2(P282S) zebrafish, further confirming the different phenotypes induced by various GABRG2 mutations.Moreover, the different sensitivity to epileptic stimuli also demonstrated the hypothesis.
9][20] In this study, they showed varied effects in the GABRG2(P282S) zebrafish.Although VPA has been used in clinics for decades, the mechanism remains elusive.
The multipotential mechanisms of VPA include blocking T-type calcium channels, 21 enhancing the expression of glutamic acid decarboxylase, promoting the release of GABA from presynaptic terminals, inhibiting GABA degradation and GABA transaminase, and increasing GABA synthesis. 22From this point of view, we speculated that the regulation of the GABAergic pathway might be an important mechanism involved in the pharmacological effect of VPA in the GABRG2(P282S) zebrafish.However, whether the modulation of protein acetylation is involved in the pharmacological effect of VPA needs further investigation.
Our previous research has found increased inflammatory factors in mutant GABRG2 transgenic zebrafish. 8Proinflammatory factors, such as IL-1β, IL-6, TNFα, and TGFβ, were reported to play important roles in epileptogenesis, [23][24][25] indicating that inflammatory response in the brain is a crucial mechanism in the pathophysiology of epilepsy. 26The activation of inflammatory response might destroy the balance between glutamate and GABA, leading to the increase of excitability in the nervous system and seizure activity. 27Here, we detected that the increase of pro-inflammatory factors was related to the epilepsy severity, more pro-inflammatory factors were found in the transgenic zebrafish showing more severe phenotype.The results suggested a positive correlation between inflammatory response and epileptic phenotype.
The comparison of brain transcriptome changes in the three zebrafish lines showed the differential activation of the IL-2 receptor signal pathway.The role of IL-2 in epilepsy is still controversial.The plasma level of IL-2 was found to be lower, 28 higher, 29 or not significantly changed 30 in epileptic patients than controls.However, these data were all collected from the patients after epileptic onset which could not exclude the influence of seizures on the expressions of cytokines.In our study, we detected differential expressions of IL-2 receptor subunits at 3 dpf even before seizure onset, indicating that cytokine-mediated inflammation might be one of the underlying mechanisms in the pathogenesis of epilepsy.IL-2 signaling pathway was activated in patients with focal cortical dysplasia (FCD), a cause of intractable epilepsy, and JAK1/3-STAT5 was the downstream of IL-2-dependent signaling pathways contributing to the pathogenesis of FCD. 31 SOCS1 was shown to be able to interact with both JAK2A and STAT5. 32Here, we also observed differential expressions of socs1, stat5a, and stat5b in the three transgenic zebrafish, which was consistent with the changes in IL-2 receptors.The results suggested that IL-2 receptor activation might be involved in epileptogenesis through SOCS1/STAT5 signaling pathway.In addition, STAT5 was also regulated by IL-1β and IL-6. 33As different expressions of proinflammatory factors and stat5 expressions were found in the three transgenic zebrafish lines, we inferred that STAT5 signaling might be one of the important pathways responsible for epileptogenesis and be related to the severity of seizure onset.
Recently, the link of the immune system to mitochondrial dynamics and morphology was reported in two patients with severe neurological deterioration following viral infection, and JAK-STAT signaling might contribute to mitochondrial disease. 34As the brain is a highly energy-demanding organ, metabolic dysfunction and mitochondrial defects contribute to acquired epilepsies, such as TLE. 35tabolic dysfunction is crucial for epigenetics because the balance of neuronal activity requires bioenergetic adaptability. 36Although there was no direct evidence suggesting the metabolic dysfunction in genetic epilepsies, some researchers have suggested that metabolism and mitochondrial defects contributing to seizure susceptibility or progression in Dravet syndrome (DS) caused by SCN1A mutations and the ketogenic diet (KD) seemed to be an effective treatment for the patients. 37,38In a zebrafish model of DS, the glycolytic and oxygen consumption rates were decreased. 39Here we observed dysregulation of the metabolic pathway in the P282S zebrafish, indicating the involvement of metabolic dysfunction in genetic epilepsy induced by GABRG2 mutations.However, the relationship between inflammatory responses and metabolic changes needs further investigation.

| CON CLUS IONS
In summary, we successfully established a transgenic zebrafish epileptic model expressing human mutant GABRG2(P282S), which was sensitive to epileptic stimulus, and the metabolic pathway was significantly changed in the brains.CBZ and VPA showed antiepileptic effects in the transgenic zebrafish.Differential inflammatory responses, especially the SOCS/JAK/STAT signaling pathway, might be related to the phenotypes of genetic epilepsy induced by GABRG2

| 3 of 14 SUI
figure legends and each sample included 10 pooled larvae.

Figure
Figure 1E).According to the epilepsy scoring system of zebrafish larvae, the activities are divided into four stages: Stage 0 (little swimming activity); Stage I (a general increase in swimming activity); Stage II (rapid "whirlpool-like" swimming behavior); Stage III (convulsions and loss of posture). 11At 5 dpf, about 30% and 66% of the WT zebrafish stayed in Stage 0 and Stage I, respectively, while only 4% reached Stage II and no zebrafish reached Stage III.On the other hand, only

SUI
et al. 6% of P282S zebrafish stayed in Stage 0. The proportion of mutant P282S zebrafish reaching Stage I (53%) and Stage II (38%) increased significantly.In addition, 3% of zebrafish reached Stage III in the P282S group (Figure 1F).
regulated) differentially expressed genes (DEGs) in P282S group F I G U R E 2 The locomotor activity changes of Tg(hGABRG2 P282S ) zebrafish with PTZ exposure and light flash stimulation.(A) The representative locomotor activity of WT and P282S transgenic zebrafish larvae at 5 dpf with 15 mM PTZ exposure.(B) The diagram showing the distance of the transgenic zebrafish traveled during the 30-min recording time after PTZ exposure.(C) The quantification of the total distance traveled for each group with PTZ exposure.**p < 0.01 by Student's t-test (n = 23 in WT and n = 21 in P282S group).(D) The mobility of the transgenic zebrafish larvae with PTZ exposure.**p < 0.01 by one-way ANOVA and subsequent Tukey's multiple comparisons (n = 23 in WT and n = 21 in P282S group).(E) The representative locomotor activity of WT and P282S transgenic zebrafish larvae measured on 5 dpf during light flash stimulation (light on for 5 s and off for 5 s, a total of 100 cycles).(F) The diagram showing the acceleration maximum of the transgenic zebrafish during the light flash stimulation.(G) The quantification of the total distance traveled for each group with light flash stimulation.*p < 0.05 by Student's t-test (n = 38 in WT and n = 33 in P282S group).(H) The mobility of the transgenic zebrafish larvae with light flash stimulation (n = 38 in WT and n = 33 in the P282S group).

F I G U R E 3
The effects of different traditional AEDs in Tg(hGABRG2 P282S ) zebrafish.(A) The representative locomotor activity of P282S transgenic zebrafish larvae treated with different traditional AEDs (100 μM CZP, 30 mM LEV, 100 μM CBZ, 100 μM VPA) for 30 min at 5 dpf.(B) The total distance of the P282S transgenic zebrafish larvae traveled with different traditional AEDs treatments.**p < 0.01 by Kruskal-Wallis test and subsequent Dunn's multiple comparisons test (n = 20 in each group).(C) Quantification of c-fos mRNA expressions in the P282S zebrafish larvae treated with different traditional AEDs at 5 dpf by qRT-PCR.The expression level in the P282S + DMSO group was set as "1" after normalization to the internal reference gene β-actin.**p < 0.01 by one-way ANOVA and subsequent Tukey's multiple comparisons (n = 6 for each group and one sample = 10 pooled larvae).(D) WISH for c-fos expression (dark purple) within the whole brain of P282S larval zebrafish treated with different traditional AEDs (100 μM CZP, 30 mM LEV, 100 μM CBZ, 100 μM VPA) for 30 min at 5 dpf.(E) The quantification of the total distance traveled with different concentrations (25, 50, 100 μM) of CBZ.**p < 0.01 by one-way ANOVA and subsequent Tukey's multiple comparisons (n = 26 in each group).(F) The quantification of the total distance traveled with different concentrations (25, 50, 100 μM) of VPA.**p < 0.01 by one-way ANOVA and subsequent Tukey's multiple comparisons (n = 20 in each group).F I G U R E 4 The comparison of behavioral activity, production of proinflammatory factors, and response to SAHA treatment in different transgenic zebrafish.(A) The total distance of the P282S, F343L, and I107T transgenic zebrafish larvae traveled with 2.5 μM SAHA treatment.**p < 0.01 by one-way ANOVA and subsequent Tukey's multiple comparisons (n = 20 in each group).(B) The response of the P282S, F343L, and I107T transgenic zebrafish larvae with 15 mM PTZ exposure.*p < 0.05, **p < 0.01 by one-way ANOVA and subsequent Tukey's multiple comparisons (n = 20 in each group).The total distance of WT zebrafish traveled with PTZ exposure was set as "1".(C) The production of IL-1β in the P282S, F343L, and I107T transgenic zebrafish larvae at 5 dpf.*p < 0.05, **p < 0.01 by one-way ANOVA and subsequent Tukey's multiple comparisons (n = 6 in each group and one sample = 10 pooled larvae).(D) The production of IL-6 in the P282S, F343L, and I107T transgenic zebrafish larvae at 5 dpf.*p < 0.05, **p < 0.01 by one-way ANOVA and subsequent Tukey's multiple comparisons (n = 6 in each group and one sample = 10 pooled larvae).(E) The production of TNFα in the P282S, F343L, and I107T transgenic zebrafish larvae at 5 dpf.**p < 0.01 by one-way ANOVA and subsequent Tukey's multiple comparisons (n = 6 in each group and one sample = 10 pooled larvae).F I G U R E 5 Brain transcriptional profile of Tg(hGABRG2 P282S ) zebrafish larvae.(A) Volcano plot of differentially expressed genes (DEGs) in the brains of P282S zebrafish and the WT controls at 3 dpf.(B) Volcano plot of DEGs in the brains of P282S zebrafish and the WT controls at 5 dpf.(C) KEGG pathway of the DEGs at 3 dpf.(D) KEGG pathway of the DEGs at 5 dpf.(E) qPCR validation of the relative expression of DEGs in the P282S zebrafish.The expression level in the WT group was set as "1" after normalization to the internal reference gene β-actin at 3 dpf and 5 dpf, respectively.*p < 0.05, **p < 0.01 by one-way ANOVA and subsequent Tukey's multiple comparisons (n = 3 for each group and one sample = 10 pooled larvae).The full list of DEGs at 3 dpf and 5 dpf is provided in Tables
mutations.Further study will expand the pathological mechanisms of genetic epilepsies and provide a theoretical basis for searching for effective drug treatment.AUTH O R CO NTR I B UTI O N S ZQ and SD designed the research.SJ and ZL conducted experiments, analyzed the data, and drafted the manuscript.JS, WW, CY, YF, LW, and WJ assisted in zebrafish breeding, cell culture, and sample collection.ZQ, SD, and CM drafted and revised the manuscript.All authors contributed to the article and approved the submitted version.FU N D I N G I N FO R M ATI O N This work was supported by the National Natural Science Foundation of China (Grant No. 82371460, 82271487, and 81771404), the Natural Science Foundation of Jiangsu Province (Grant No. BK20201440), the Postgraduate Research & Practice Innovation